The emergence and rapid biogeographic expansion of West Nile Virus (WNV; Flaviviridae; Flavivirus) throughout the western hemisphere, as well as the re-emergence of other medically significant viruses, highlight the vulnerability of the United States to both novel and reemerging viral pathogens. To understand how zoonotic viruses evolve following introduction to new habitats, it is necessary to address the selective pressures shaping viral adaptation. Specifically, we must characterize factors important in determining the plasticity (ability of individual genotypes to generalize to many hosts) and adaptability (ability to rapidly produce fitter genotypes) of virus populations interacting with different hosts in order to evaluate the capacity for geographic expansion into new environments. The mechanisms that account for success of a virus in colonization of new environments are poorly characterized. Experimental studies in naturally relevant hosts are generally lacking in virus adaptation studies. These studies are required to provide plausible and relevant mechanistic explanations for adaptability. The proposed research will address this fundamental gap in the state of knowledge through laboratory studies using natural and ecologically relevant mosquito and avian hosts. Studies in the laboratory are the best way to conduct a systematic evaluation of the factors that influence viral adaptation. We have chosen to undertake a comparative study with two flaviviruses, WNV and St. Louis encephalitis virus (SLEV). These two viruses are very closely related antigenically, genetically, and structurally. Nevertheless, their epidemiological patterns in the western hemisphere have differed strongly in recent years. WNV has continued to expand both its host and geographic ranges, producing significant levels of disease in the U.S. every year since its emergence. SLEV, on the other hand, exhibits only periodic outbreaks which remain relatively contained. Whether WNV activity in North America will ultimately mimic that of SLEV remains to be seen, but a comparative study of current genotypes should elucidate genetic correlates which define variable epidemiological patterns. Recent experimental studies in our laboratory have revealed apparent differences in in vitro adaptation between SLEV and WNV. Our extensive BSL-3 laboratory, insectary, and vivarium will allow us to confirm and expand the in vitro results in vivo in natural hosts and further characterize important factors that both constrain and facilitate arbovirus activity in disparate hosts and new environments. These are among the first studies, to the best of our knowledge, using natural vertebrate and mosquito hosts to elucidate mechanisms of viral adaptation.